| Summary: | 碩士 === 國立臺灣大學 === 化學研究所 === 100 === Protein detection is of great importance in basic research and clinical diagnosis of genetic disorders and its associated diseases, cancers, and pathogen infections. We have developed two colorimetric protein sensors using aptamer modified 13-nm gold nanoparticles (Apt–Au NPs), thrombin, and fibrinogen adsorbed Au NPs (Fib–Au NPs; 56 nm). These could be used for the highly selective and sensitive detection of platelet-derived growth factors (PDGFs) and human immunoglobulin G (hIgG). In the PDGF system, Apt–Au NPs and Fib–Au NPs were the recognition and reporting units, respectively. PDGF-binding-aptamer (AptPDGF) and 29-base-long thrombin-binding-aptamer (Aptthr29) were conjugated with Au NPs to prepare functional Apt–Au NPs (AptPDGF/Aptthr29–Au NPs) for specific interaction with PDGF and thrombin, respectively. Thrombin interacted with Fib–Au NPs in solution to catalyze the formation of insoluble fibrillar fibrin–Au NPs agglutinates through the polymerization of unconjugated and conjugated fibrinogen. Thrombin activity was suppressed when it interacted with AptPDGF/Aptthr29–Au NPs due to steric effects through the specific interaction of PDGF with AptPDGF on the surfaces of AptPDGF/Aptthr29–Au NPs. Under optimal conditions with AptPDGF/Aptthr29–Au NPs at 25 pM, thrombin at 400 pM, and Fib–Au NPs at 30 pM, AptPDGF/Aptthr29–Au NPs/Fib–Au NPs probe responded linearly to PDGF over a concentration range of 0.5–20 nM with a correlation coefficient of 0.96. The limit of detection (LOD, signal-to-noise ratio = 3) for each of the three PDGF isoforms was 0.3 nM in the presence of bovine serum albumin at 100 μM. When using AptPDGF/Aptthr29–Au NPs to selectively enrich PDGF and remove interfering substances from cell media, LOD of this probe for PDGF was 35 pM. This probe revealed that the concentration of PDGF in the three cell media is 230 (±20) pM, showing its advantages in terms of simplicity, sensitivity, and specificity. We also developed a method for the selective
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and sensitive detection of human immunoglobulin G (hIgG). The first step involves the specific interactions of hIgG with protein G (PG)-functional Fe2O3 magnetic NPs (PG–MNPs) and with PG– and aptamer (Apt)– modified gold NPs (PGApt–Au NPs) and the subsequent magnetic separation of their complexes (PG–MNPs…hIgG…PGApt–Au NPs). In the second step, the concentration of free PGApt–Au NPs was determined by taking advantage of their control of thrombin activity toward fibrinogen-modified Au NPs (Fib–Au NPs). The activity of thrombin toward Fib–Au NPs to form fibrin–Au NP aggregates was inhibited by PGApt–Au NPs through the specific interaction of thrombin with the Apt. The greater the amount of hIgG in a sample, the less free PGApt–Au NPs remained in the supernatant. Consequently, greater amounts of free thrombin remained, which led to the formation of greater amounts of fibrin–Au NP aggregates. Under optimal conditions (8 μg/mL PG–MNPs, 1.0 nM PGApt–Au NPs, 400 pM thrombin, 30 pM Fib–Au NPs), PG–MNPs/PGApt–Au NPs/Fib–Au NPs probe allows the selective detection of hIgG down to 5 nM in the presence of 100 μM of BSA. The practicality of this approach was validated by determining the concentrations of hIgG in spiked plasma samples that were in good agreement with determinations made by enzyme-linked immunosorbent assays (R2 = 0.98). These results demonstrate that this assay has great potential for diagnosing diseases associated with changes in hIgG levels.
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